The present invention relates to specific proteins as well as recombinant versions of these proteins which are organic anion transporters. These proteins include human membrane proteins. preferentially found in kidney and liver cells. The present invention also relates to nucleotide sequences encoding these novel organic anion transporters. In another aspect, the present invention relates to methods for using these proteins as in vitro screening agents to identify substrates for the proteins and inhibitors which block transport activity. In yet another aspect, these proteins may be used in in vitro assays to predict drug pharmacokinetics, drug distribution and drug toxicity. The invention also discloses antibodies that specifically bind to these proteins and which may be used in diagnostic assays.
The following describes certain relevant art, none of which is admitted to be prior art to the inventions described herein.
The liver and kidney are two organs that can extract a variety of organic anions from circulation, including endogenous compounds such as bile acids and bilirubin and xenobiotics such as sulfobromophthalein (BSP) and p-aminohippurate (PAH). The liver and kidney thus have the critical functions of bile secretion, detoxification, and drug elimination. Since the cell membrane represents a hydrophobic barrier that prevents influx of charged or hydrophilic molecules, hepatocytes and kidney epithelial cells express proteins on their basolateral membranes to facilitate transport of organic anions into the cell. The liver and kidney contain membrane proteins that transport very specific compounds such as taurocholic acid or prostaglandins. However, they also have proteins that exhibit a wider substrate specificity. Recently, transporters of the latter type have been cloned from rat liver and kidney. One such protein is called the organic anion transporting polypeptide, oatp (Jacquemin et al. (1994) PNAS 91:133-37). This protein belongs to a family of related transporters, members of which consists of oatp1, oatp2, oatp3, and prostaglandin transporters. These proteins share homology at the amino acid level (Noe et al. (1997) PNAS 94: 10346-50; Kanai et al. (1995) Science 268: 866-69; Abe et al. (1998) J. Biol. Chem. 273: 22395-401). When expressed in Xenopus oocytes or in mammalian cells, members of the oatp family were shown to transport many types of organic anions including taurocholic acid, BSP, and conjugated steroid hormones. The related prostaglandin transporters show high affinity transport of prostaglandins.
Shortly after the cloning of oatp1, members of another organic anion transporter family, OAT1 and OAT2, were isolated from rat kidney and liver, respectively (Sekine et al. (1997) J. Biol. Chem 272: 18526-29; Sekine et al. (1998) FEBS Letters 429: 179-82). Rat OAT1 and OAT2 are homologous to each other but show no significant homology to members of the oatp family. However, they show some homology to the family of organic cation transporters (OCTs), suggesting that these two families of proteins share a common origin. Rat OAT1 and OAT2 can transport many different organic anions. OAT1, when expressed in oocytes, can transport p-aminohippurate (PAH), methotrexate, and glutarate. OAT1 is most likely the molecular entity responsible for the classical p-aminohippurate/xcex1-ketoglutarate exchanger found in the kidney proximal tubule. OAT2, expressed predominantly in the liver, has been shown to transport organic anions such as salicylate, methotrexate, and xcex1-ketoglutarate. Therefore, OAT1 and OAT2 belong to yet another family of multi-specific organic anion transporters distinct from the oatp family.
The present invention is directed to a group of polypeptides, preferentially expressed in the liver and kidneys of humans and which have activity as organic anion transporters. We refer to these polypeptides as xe2x80x9chuman organic anion transportersxe2x80x9d or xe2x80x9chOAT polypeptidesxe2x80x9d. These proteins and their properties are described in detail below.
The invention also concerns, nucleic acids encoding hOAT polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to the polypeptides, assays utilizing the polypeptides, and methods relating to all of the foregoing.
A first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding an hOAT polypeptide or encoding a fragment of an hOAT polypeptide.
In preferred embodiments the isolated nucleic acid comprises a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, a nucleic acid sequence that hybridizes to the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof.
The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester or other method or by using an automated DNA synthesizer. Preferably, the nucleic acid is isolated from mammalian kidney or brain, most preferably from liver.
Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
In another preferred embodiment, the nucleic acid molecules of the invention comprise nucleotide sequences that (a) have the nucleic acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, or encode polypeptides having the full-length amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a functional derivative thereof, (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecules of (a) and encodes a naturally occurring hOAT polypeptide; (d) encodes an hOAT polypeptide having the full-length amino acid sequence of the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 except that it lacks one or more of the domains selected from the group consisting of an extracellular domain, a transmembrane domain, and an intracellular domain; or (e) is the complement of the nucleotide sequence of (d).
The nucleic acid molecules of the invention are isolated, enriched, or purified preferably from a mammal, more preferably from a human.
Hybridization techniques may be used to isolate a nucleic acid molecule of interest. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Highly stringent conditions may mean conditions that are at least as stringent as the following: hybridization in 50% formamide, 5xc3x97SSC, 50 mM NaH3PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5xc3x97Denhart solution at 42xc2x0 C. overnight; washing with 2xc3x97SSC, 0.1% SDS at 45xc2x0 C.; and washing with 0.2xc3x97SSC, 0.1% SDS at 45xc2x0 C. Those skilled in the art will recognize how such conditions can be varied to vary specificity and selectivity.
Particularly preferred embodiments of this aspect of the invention are naturally or non-naturally occurring variants of the nucleic acids of the invention. Among variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Such nucleic acid molecules are identifiable as being able to hybridize to or which are at least about 60-65% percent identical, preferably at least about 70-75% percent identical, more preferably at least about 80-83% percent identical, and even more preferably at least about 87-95% percent identical to the nucleic acid molecules shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
Among the particularly preferred embodiments of the invention in this regard are nucleic acid molecules encoding polypeptides having the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12; variants, analogs, derivatives and fragments thereof, and fragments of the variants, analogs and derivatives.
Further particularly preferred in this regard are nucleic acid molecules encoding OAT variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, which have the amino acid sequence for the hOAT polypeptides of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the OAT polypeptides. Also especially preferred in this regard are conservative substitutions. In one embodiment, the polypeptide encoded by the nucleic acid molecule is at least about 30-35%, preferably at least about 40-45%, more preferably at least about 50-55%, even more preferably at least about 60-65%, yet more preferably at least about 70-75%, still more preferably at least about 80-85%, and most preferably at least about 90-95% or more identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
In still another embodiment, the nucleic acid molecule encodes a naturally occurring variant of the polypeptide of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 and hybridizes under stringent conditions to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, respectively.
Further preferred embodiments are variants of the herein described nucleic acid molecules in which the transporting activity of the polypeptides is altered. Such alterations can result in either increased, or decreased transport of substrates recognized by hOAT polypeptides. Further, variants can be made that result in OAT polypeptides which are no longer responsive to cellular signals, for example, protein kinase C deactivation of transport.
Especially preferred are nucleic acid molecules encoding OATs which are expressed on the basolateral (sinusoid) membrane of hepatocyte cells.
Included in the invention are all possible nucleic acid sequences that encode the amino acid sequences of the hOAT polypeptides of the invention. As it is recognized that alternate codons will encode the same amino acid for most amino acids due to the degeneracy of the genetic code, the sequences of this aspect includes nucleic acid sequences utilizing such alternate codon usage for one or more codons of a coding sequence. For example, all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acid, alanine. Therefore, if for an amino acid there exists an average of three codons, a polypeptide of 100 amino acids in length will, on average, be encoded by 3100, or 5xc3x971047, nucleic acid sequences. Thus, a nucleic acid sequence can be modified (e.g., a nucleic acid sequence from an hOAT as specified above) to form a second nucleic acid sequence encoding the same polypeptide as encoded by the first nucleic acid sequence using routine procedures and without undue experimentation. Thus, all possible nucleic acid sequences that encode the amino acid sequences of the hOAT polypeptides of the invention are included in the present invention and are described, as if all were written out in full, taking into account the codon usage.
The alternate codon descriptions are available in common textbooks, for example, Stryer, BIOCHEMISTRY 3rd ed., and Lehninger, BIOCHEMISTRY 3rd ed. Codon preference tables for various types of organisms are available in the literature. Because of the number of sequence variations involving alternate codon usage, for the sake of brevity, individual sequences are not separately listed herein. Instead the alternate sequences are described by reference to the natural sequence with replacement of one or more (up to all) of the degenerate codons with alternate codons from the alternate codon table, preferably with selection according to preferred codon usage for the normal host organism or a host organism in which a sequence is intended to be expressed. Those skilled in the art also understand how to alter the alternate codons to be used for expression in organisms where certain codons code differently than shown in the xe2x80x9cuniversalxe2x80x9d codon table.
In a second aspect, the invention relates to a nucleic acid vector comprising a nucleic acid molecule encoding an hOAT polypeptide and a promoter element effective to initiate transcription in a host cell.
Those skilled in the art would recognize that a nucleic acid vector can contain many other nucleic acid elements besides the promoter element and the hOAT nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, periplasm or peroxisome localization signals, or signals useful for polypeptide purification.
A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that the restriction enzymes operate upon are readily available to those skilled in the art. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
Many techniques are available to those skilled in the art to facilitate transformation or transfection of the expression construct into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest.
In a third aspect, the invention features a nucleic acid probe for the detection of a nucleic acid encoding an hOAT polypeptide, fragment or analogue in a sample. The nucleic acid probe contains nucleotides that will hybridize specifically to a sequence of at least 14 contiguous nucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID. NO:5, or SEQ ID NO:6, or a functional derivative thereof. The probe is preferably at least 14 or more bases in length and selected to hybridize specifically to a unique region of an hOAT encoding nucleic acid.
In preferred embodiments, the nucleic acid probe hybridizes to at least 14 nucleotides of a nucleic acid encoding the full-length sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a functional derivative thereof. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under highly stringent hybridization conditions, only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
In another preferred embodiment, the nucleic acid is an isolated conserved or unique region, for example, those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, or for the design of PCR probes to facilitate amplification or cloning of additional polypeptides. Such a nucleic acid may contain additional sequences in addition to the conserved or unique region, preferably 10-20 additional nucleotides on each side of the conserved or unique region, more preferably 30-40 additional nucleotides, and most preferably 75-100 additional nucleotides.
Methods for using the probes include detecting the presence or amount of hOAT polypeptide RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to hOAT polypeptide RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for an hOAT polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container having disposed therein a nucleic acid probe.
Another feature of the invention is a nucleic acid molecule as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or fragments thereof, comprising one or more regions that encode an hOAT polypeptide or an hOAT domain polypeptide, where the hOAT polypeptide or the hOAT domain polypeptide is fused to a non-hOAT polypeptide or amino-terminal tag. Possible non-hOAT polypeptide or amino-terminal tag fusion partners include, for example, but are not limited to, glutathione-S-transferase (GST)-fusion proteins, green fluorescent protein (GFP) and fusions with histidine residues as described by Janknecht et al., 1991, Proc. Natl. Sci. USA 88:8972-8976.
The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding an hOAT polypeptide and a transcriptional termination region functional in a cell.
Another aspect of the invention relates to a recombinant cell or tissue comprising a nucleic acid molecule encoding an hOAT polypeptide or hOAT polypeptide fragment. The recombinant cell may comprise a nucleic acid molecule encoding an hOAT polypeptide; an hOAT domain polypeptide; an hOAT polypeptide fragment or an hOAT polypeptide or polypeptide fragment fused to a non-hOAT polypeptide. The recombinant cell can harbor a nucleic acid vector that is extragenomic or intragenomic.
Extragenomic vectors are designed with their own origins of replication allowing them to utilize the recombinant cell replication machinery to copy and propagate the vector nucleic acid sequence. These vectors are small enough that they are not likely to harbor nucleic acid sequences homologous to genomic sequences of the recombinant cell.
Multiple intragenomic vectors are available to those skilled in the art and contain nucleic acid sequences that are homologous to nucleic acid sequences in a particular organism""s genomic DNA. These homologous sequences will result in recombination events that integrate portions of the vector into the genomic DNA. Those skilled in the art can control which nucleic acid sequences of the vector are integrated into the cell genome by flanking the portion to be incorporated into the genome with homologous sequences in the vector.
A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
In yet another aspect, the invention features an isolated, enriched, or purified hOAT polypeptide.
It is also advantageous for some purposes that an amino acid sequence be in purified form. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure. The hOAT polypeptides, or fragments thereof, of the present invention are preferably isolated, purified, or enriched from a mammal or a mammalian cell. The mammal is as defined herein and preferably is a mouse, and most preferably is a human. These polypeptides may be isolated, purified, or enriched from a cell that comprises an endogenous nucleic acid molecule that encodes the polypeptide, or from a cell that is transformed with a nucleic acid molecule that encodes the polypeptide. The polypeptide may also be chemically synthesized. Procedures for obtaining polypeptides using the above methods are well known to those skilled in the art.
The polypeptides of the invention comprise an amino acid sequence having (a) the full-length amino acid sequence encoded by the nucleic acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or functional derivatives thereof, or the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or functional derivatives thereof.
The invention also features an isolated, enriched, or purified hOAT polypeptide fragment.
Examples of polypeptide fragments include hOAT domains, and hOAT-specific epitopes. Such fragments may, for example, be produced by proteolytic cleavage of the full-length protein. Preferably, the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence. Fragments of a protein can be used as antigens for the production of hOAT-specific antibodies, or used as competitors of substrates for hOAT polypeptides. It is understood that such fragments may retain one or more characterizing portions of the native complex. Examples of such retained characteristics include: transporting activity; substrate specificity; interaction with other molecules in the intact cell; regulatory functions; or binding with an antibody specific for the native complex, or an epitope thereof.
Well-known examples of domains are the SH2 (Src Homology 2) domain (Sadowski, et al., (1986) Mol. Cell. Biol. 6:4396; Pawson and Schlessinger, (1993) Curr. Biol. 3:434), the SH3 domain (Mayer, et al., (1988) Nature 332:272; Pawson and Schlessinger, (1993) Curr. Biol. 3:434), and pleckstrin (PH) domain (Ponting, (1996) TIBS 21:245; Hasiam, et al., (1993) Nature 363:309), all of which are domains that mediate protein:protein interaction, and the kinase catalytic domain (Hanks and Hunter, (1995) FASEB J 9:576-595). The relative homology is at least 20%, more preferably at least 30% and most preferably at least 35%. Computer programs designed to detect such homologies are well known in the art.
Comparisons between the sequences of two or more polynucleotides or polypeptides can be performed using the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, or the method of Pearson and Lipman, (1988) PNAS 85:2444. Computer programs implementing these methods can be used and include, BLAST, GAP, BESTFIT, FASTA, and TFASTA which are offered in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.
Protein motifs can be identified using computer programs readily available to those skilled in the art. One such computer program is offered by the Institute for Chemical Research at Kyoto University. The Kyoto computer program is offered at the following world wide web site (motif.genome.ad.jp/).
Protein topology, such as the orientation and location of transmembrane helixes can also be identified using readily available computer programs. One such program is TopPred2 offered by Stockholm University and offered at the following world wide web site (biokemi.su.se/xcx9cserver/toppred2/).
Another aspect of the invention features an isolated, enriched or purified hOAT polypeptide analog.
The hOAT polypeptide analogs of the present invention preferably have a substantially similar biological activity to the proteins encoded by the full-length nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or the amino acid sequence set forth in SEQ ID NO:7, or SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
A sequence that is substantially similar will preferably have at least 85% identity (more preferably at least 90% and most preferably 95-100%) to the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ. ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
An hOAT polypeptide analog will retain some useful function such as, for example, substrate binding, transport activity, or the ability to bind to an hOAT specific antibody (as defined below). The hOAT polypeptide analog may be derived from a naturally occurring complex component by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence. It is understood that such analogs having added, substituted and/or additional amino acids retain one or more characterizing portions of the native hOAT polypeptides.
Examples of hOAT polypeptides analogs include, but are not limited to, (1) chimeric proteins which comprise a portion of an hOAT polypeptide sequence fused to a non-hOAT polypeptide sequence, for example, a polypeptide sequence of glutathione-S-transferase, (2) hOAT polypeptides lacking a specific domain, for example, the protein kinase C domain, (3) epitope-tagged hOAT polypeptides or fragments for immuno detection or purification and (4) hOAT proteins having a point mutation.
Human OAT polypeptide analogs with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well known to those of ordinary skill in the art. For example, the modified components of the analogs may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al., (1983) DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is produced, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above. Alternatively, proteins with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well known in the art. The analogs of the hOAT polypeptides may exhibit the same qualitative biological activity as the hOAT polypeptides themselves.
In another aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific-binding affinity to an hOAT polypeptide or hOAT polypeptide analog or fragment.
Antibodies having specific-binding affinity to an hOAT polypeptide may be used in methods for detecting the presence and/or amount of an hOAT polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the hOAT polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
Monoclonal antibodies may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., (1975) Nature 256:495-497, and U.S. Pat. No. 4,376,110 issued Mar. 8, 1983, to David et al.
Still further, the invention features a hybridoma which produces an antibody having specific-binding affinity to an hOAT polypeptide.
In preferred embodiments, the hOAT antibody comprises a sequence of amino acids that is able to specifically bind an hOAT polypeptide.
The invention features a method for identifying human cells containing an hOAT polypeptide or a related sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying hOAT (eg., cloning, Southern or Northern blot analysis, Western blot analysis, immunoassay, in situ hybridization, PCR amplification, etc.).
The invention also features methods of screening cells for natural-binding partners of hOAT polypeptides. Binding partners include modulators and downstream signaling molecules such as adaptor proteins and may be identified by techniques well known in the art such as co-immunoprecipitation or by using, for example, a two-hybrid screen. (Fields and Song, U.S. Pat. No. 5,283,173, issued Feb. 1, 1994, and incorporated by reference herein). The present invention also features the purified, isolated or enriched versions of the polypeptides identified by the methods described above.
The invention provides methods for screening compounds for their ability to inhibit, or modulate the biological activity of the human organic anion transporter molecules of the invention. In preferred embodiments, cells expressing an hOAT polypeptide, including recombinant expression constructs of the invention, are contacted with a compound, and transport activity is assayed. In especially preferred embodiments, competition assays, using known human organic anion transporter substrates, such as p-aminohippurate, are used to test the ability of a screened compound to interfere (compete) with the uptake of a known substrate of an hOAT polypeptide. Additional preferred embodiments comprise quantitative analyses of such effects.
The present invention is also useful for the detection of substrates, analogues of substrates, inhibitors and modulators, heretofore known or unknown, for the transporters of the invention, either naturally occurring or embodied as a drug. In preferred embodiments, such substrates, analogues, or modulators may be detected in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, vitreous humor, or any other bodily fluid. In additional preferred embodiments, the invention provides methods for detecting and identifying substrates, analogues of substrates, or modulators that preferentially affect either the uptake function or the efflux function of the transporters of the invention.
One method for identifying a substance capable of modulating an hOAT polypeptide activity comprises the steps of (a) contacting an hOAT polypeptide with a test substance; and (b) determining whether the substance alters the transporting activity of said polypeptide.
Another method of identifying substances capable of modulating the function of an hOAT polypeptide comprises the following steps: (a) expressing an hOAT polypeptide in cells; (b) adding a compound to the cells; and (c) monitoring the transporting activity of the hOAT.
A method of identifying substrates or inhibitors of hOAT polypeptides comprises the following steps: (a) adding a test substrate to a cell line expressing an hOAT polypeptide; and (b) measuring the intracellular concentration of the test substrate.
A second method of identifying substrates or inhibitors of hOAT polypeptides comprises the following steps: (a) adding a test substrate to a cell line expressing an hOAT polypeptide; (b) adding a substrate known to be transported by said hOAT polypeptides; (c) measuring whether said test substrate competes with the uptake of the known substrate.
Another method of identifying substrates of an hOAT polypeptide comprises the following steps: (a) attaching a fluorescent compound onto a test compound; (b) adding said compound to a cell line expressing an hOAT polypeptide; and (c) monitoring whether said fluorescent compound is taken into said cell line through the use of fluorescent microscopy or any other method which can detect fluorescence.
In a preferred embodiment, high-throughput screening employing 96-well plates and a microtiter(copyright) fluorescence detection system is used to detect potential modulators, substrates, analogues of substrates and inhibitors of hOAT polypeptides which can compete with binding or transport of fluorescent compounds known to bind or be transported by hOAT polypeptides.
In yet another embodiment, uptake of a test substrate by an hOAT expressing cell line is measured using knowledge of an hOAT""s antiporter activity. Uptake is measured by monitoring efflux of an accompanying antiporter molecule. Efflux of the antiporter molecule can be detected using techniques known to those skilled in the art, for example, radiolabeling of the effluxed molecule and measurement using a scintillation counter. Measurement of antiporter efflux,is also amenable to high-throughput screening.
A still further aspect of the invention is the identification of substrates, analogues of substrates, inhibitors, or modulators that affect the pharmacokinetics of drugs and compounds transported by hOAT polypeptides. Substances which decrease the transporting activity of hOAT polypeptides are useful for increasing the half-life in the body of drugs and compounds excreted via human organic anion transporters.
Yet, another aspect of the invention involves substrates, and modulators, which increase the transporting activity of an hOAT. Such substances are advantageous for increasing the delivery of a drug or compound, which is transported by an hOAT, to a target organ such as kidney or liver. For example, a modulator which increases the transporting activity of hOAT1 would increase the delivery of PAH or other drugs or compounds transported by hOAT1 to the kidney.
A last aspect of the invention features a cultured cell line which stably expresses an hOAT polypeptide. Such cell lines are useful for identifying substrates which are transported by hOAT polypeptides, inhibitors of hOAT polypeptides and for identifying modulators of hOAT polypeptides. Further, a panel of cells stably expressing hOAT polypeptides will be useful in studying the pharmacokinetics of various drugs and compounds. Preferably, the expression of an hOAT polypeptide in such cell lines is under the control of an inducible promoter. More preferably, said inducible promoter has a very low basal level of expression in such cell lines and high expression when induced.
A cell line stably expressing an hOAT polypeptide is useful for designing substrates which can be transported by an hOAT polypeptide at either an increased or a decreased rate. Decreasing the rate of transport can increase the effectiveness of some medicinals by making their clearance from the body occur more slowly.
Cell lines stably expressing hOAT polypeptides are also useful for the identification and development of drugs or compounds targeted to a particular organ. For example, drugs or compounds identified as substrates of hOAT1 would be good candidates for treating diseases of the kidneys whereas those identified as substrates of hOAT2 would be good candidates for treating diseases of the liver.
The OAT expressing cell lines are also useful for identifying potentially toxic compounds or drugs. For example, newly discovered or modified drugs belonging to a class of drugs known to be toxic to certain organs, e.g., kidney can be tested for transport against the panel of cells expressing human OAT1 or hOAT3 polypeptides. If the new drug or compound is transported by hOAT1 expressing cells, the new drug or compound may also be toxic to the kidney. However, if the new drug or compound, belonging to a class known to be toxic, is not transported by hOAT1 or hOAT3 expressing cells than the new drug or compound would be expected to be less toxic to the kidney than the parent class of drugs or compounds.
A preferred embodiment includes the identification of toxic compounds which are substrates of hOATs, and which can be used to treat diseases of organs or tissues that express hOATs. For example, toxic compounds found to be transported by hOAT1 or hOAT3 would have potential use as anti-cancer drugs to treat cancers of the kidney.
The methods of the present invention can utilize any of the molecules disclosed in the invention. These molecules include nucleic acid molecules encoding hOAT polypeptides, nucleic acid vectors, recombinant cells, polypeptides, or antibodies described herein.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Definitions
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid it is meant a polymer preferably consisting of 14 or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide sequence present, but that it is essentially free (about 90-95% pure at least) of nucleotide material naturally associated with it and thus is meant to be distinguished from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
The term xe2x80x9csignificantxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes the sequence from naturally occurring enrichment events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). The term is also chosen to distinguish clones already in existence which may encode an hOAT polypeptide but which have not been isolated from other clones in a library of clones. Thus, the term covers clones encoding an hOAT polypeptide which are isolated from other non-hOAT clones.
The term xe2x80x9cnucleic acid moleculexe2x80x9d describes a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
The term xe2x80x9ccDNA cloningxe2x80x9d refers to hybridizing a small nucleic acid molecule, a probe, to cDNA. The probe hybridizes (binds) to complementary sequences of cDNA.
The term xe2x80x9ccomplementaryxe2x80x9d describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a xe2x80x9ccomplementxe2x80x9d of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
The term xe2x80x9chybridizexe2x80x9d refers to a method of interacting a nucleic acid sequence with a DNA or RNA molecule in solution or on a solid support, such as cellulose or nitrocellulose. If a nucleic acid sequence binds to the DNA or RNA molecule with high affinity, it is said to xe2x80x9chybridizexe2x80x9d to the DNA or RNA molecule. The strength of the interaction between the probing sequence and its target can be assessed by varying the stringency of the hybridization conditions. Under highly stringent hybridization conditions, only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or two mismatches out of 20 contiguous nucleotides.
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, it is meant regions present on two or more nucleic acids encoding an hOAT polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acids encoding hOAT polypeptides are provided in Abe, et al. (1992) J. Biol. Chem. 19:13361 (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 contiguous nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d is meant a sequence present in a full-length nucleic acid coding for an hOAT polypeptide that is not present in a sequence coding for any other known naturally occurring polypeptide. Such regions preferably comprise 12 or more contiguous nucleotides present in the full-length nucleic acid encoding an hOAT polypeptide. In particular, a unique nucleic acid region is preferably of human origin.
The term xe2x80x9cnucleic acid vectorxe2x80x9d relates to a single or double stranded circular nucleic acid molecule that can be transfected or transformed into cells and replicate independently or within the host cell genome.
The terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d refer to methods of inserting an expression construct into a cellular organism.
The term xe2x80x9cpromoter elementxe2x80x9d describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. The promoter element precedes the 5xe2x80x2 end of the hOAT polypeptide nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
By an xe2x80x9chOAT polypeptidexe2x80x9d is meant the full-length amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a functional derivative thereof.
The term xe2x80x9crecombinant cellxe2x80x9d refers to a cell that has been altered to contain a new combination of genes or nucleic acid molecules.
The term xe2x80x9crecombinant organismxe2x80x9d refers to an organism that has a new combination of genes or nucleic acid molecules.
The term xe2x80x9corganismxe2x80x9d relates to any living being comprised of at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, a recombinant organism can also be a recombinant cell, which may be a eukaryotic or a prokaryotic organism.
By xe2x80x9crecombinant hOAT polypeptidexe2x80x9d it is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
The term xe2x80x9ceukaryotexe2x80x9d refers to an organism comprised of cells that contain a nucleus. Eukaryotes are differentiated from xe2x80x9cprokaryotesxe2x80x9d which do not have a nucleus and lack other cellular structures found in eukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotes include unicellular organisms, such as bacteria, while eukaryotes are represented by yeast, invertebrates, and vertebrates.
The term xe2x80x9cextragenomicxe2x80x9d refers to a nucleic acid vector which does not insert into the cell genome. Thus, these vectors replicate independently of the host genome and do not recombine with or integrate into the genome.
The term xe2x80x9cintragenomicxe2x80x9d defines a nucleic acid construct that is incorporated within the cell genome.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 6, 12, 18 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide it is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
The term xe2x80x9csignificantxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there are no amino acid sequences from other sources. The other source amino acid sequences may, for example, comprise amino acid sequences encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired amino acid sequences.
The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL).
By xe2x80x9can hOAT polypeptide fragmentxe2x80x9d it is meant an amino acid sequence that is less than the full-length hOAT amino acid sequences (and which excludes the listed EST sequences) encoded by the nucleic acid sequence set forth in SEQ ID NO:1 (excluding EST clone R25797), SEQ ID NO:2 (excluding EST clone A1016020), SEQ ID NO:3 (excluding EST clone A 1016020), SEQ ID NO:4 (excluding EST clone H41333), SEQ ID NO:5, or SEQ ID NO:6 (excluding EST clone AA705512) or the amino acid sequence (excluding the amino acids encoded by the EST clones previously listed set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Thus, the term xe2x80x9cfragmentxe2x80x9d is used to indicate a polypeptide derived from the amino acid sequence of the hOAT polypeptides, of the complexes having a length less than the full-length polypeptide from which it has been derived.
By xe2x80x9can hOAT domainxe2x80x9d it is meant a portion of the hOAT polypeptide having homology to amino acid sequences from one or more known proteins wherein the sequence predicts some common function, interaction or activity.
By xe2x80x9chOAT polypeptide analogxe2x80x9d it is meant an amino acid sequence substantially similar to the sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or the amino acid sequence set forth in SEQ ID NO:7, or SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
By xe2x80x9cbiological activityxe2x80x9d it is meant an activity of the hOAT polypeptides in a cell. The biological activity of the hOAT polypeptides is related to some of the activities of the cell which include, but are not limited to, transport (uptake or excretion of a substrate from a cell), cell proliferation, mitogenesis, metastasis, tumor escape, cell adhesion, transformation, or apoptosis.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
An hOAT polypeptide analog may differ from the native sequence of an hOAT polypeptide in that one or more amino acids have been changed, added or deleted. Changes in amino acids may be conservative or non-conservative. By xe2x80x9cconservativexe2x80x9d it is meant the substitution of an amino acid for one with similar properties such as charge, hydrophobicity, structure, etc.
By xe2x80x9chOAT-specific epitopexe2x80x9d it is meant a sequence of amino acids that is both antigenic and unique to hOAT.
By xe2x80x9cspecific-binding affinityxe2x80x9d is meant that the antibody binds to target (hOAT) polypeptides with greater affinity than it binds to other polypeptides under specified conditions.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody, for example an hOAT antibody.
By xe2x80x9cnatural-binding partnerxe2x80x9d it is meant a protein, organic anion, or other molecule that interacts with an hOAT polypeptide.
As used herein, pharmacokinetics is the process by which a drug or compound is absorbed, distributed, metabolized and eliminated by the body.
The term xe2x80x9ccompoundxe2x80x9d includes small organic or inorganic molecules of molecular weight of preferably less than 1000 atomic units, more preferably less than 800 atomic units, and most preferably less than 500 atomic units. xe2x80x9cOrganic moleculesxe2x80x9d include all molecules that contain a carbon atom, whereas xe2x80x9cinorganic moleculesxe2x80x9d are those that do not have a carbon atom.
The term xe2x80x9cfunctionxe2x80x9d refers to the cellular role of a protein. The role of the proteins of the invention may include transport of substrates into and out of a cell, involvement in cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.
The term xe2x80x9cmodulatesxe2x80x9d refers to the ability of a compound to alter the function of an hOAT polypeptide. A modulator preferably activates or decreases the transporter activity of an hOAT protein. A modulator that increases the transporting activity is appositive modulator; and one that decreases the transporting activity is a negative modulator. The term xe2x80x9cmodulatesxe2x80x9d also refers to altering the function of a protein by increasing or decreasing the probability that a complex, i.e. an assembly of at least two molecules bound to one another, forms between an hOAT protein and a natural-binding partner.
The term xe2x80x9ctransporting activity,xe2x80x9d in the context of the invention, defines the ability of a transporter polypeptide to uptake a substrate into a cell or efflux a molecule out of a cell.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a molecule that is transported into or out of a cell by an OAT polypeptide. The substrate may be an organic compound or molecule, inorganic compound or molecule, a peptide, or a protein.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the transport or efflux of a molecule into or out of a cell.
The term xe2x80x9cinhibitorxe2x80x9d refers to a compound or substance that binds to a substrate-binding site and, thereby, decreases or prevents transport of an hOAT substrate.
The term xe2x80x9cexpressingxe2x80x9d as used herein refers to the production of an hOAT polypeptide from a nucleic acid vector containing an hOAT gene within a cell.
The term xe2x80x9caddingxe2x80x9d as used herein refers to administering a solution comprising a compound to the medium bathing cells.
The term xe2x80x9cfunctional derivativexe2x80x9d with respect to a polypeptide is a polypeptide that possesses a biological activity (either functional or structural) or an immunological characteristic that is substantially similar to a biological activity or an immunological characteristic of a non-recombinant hOAT. A functional derivative of an hOAT polypeptide may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term xe2x80x9cfunctional derivativexe2x80x9d is intended to include the xe2x80x9cfragmentsxe2x80x9d, xe2x80x9cvariantsxe2x80x9d, xe2x80x9canaloguesxe2x80x9d, xe2x80x9chomologuesxe2x80x9d or chemical derivatives of a molecule.
Similarly, a xe2x80x9cfunctional derivativexe2x80x9d of a gene encoding an hOAT polypeptide of the present invention includes xe2x80x9cfragmentsxe2x80x9d, xe2x80x9cvariantsxe2x80x9d, or xe2x80x9canaloguesxe2x80x9d of the gene, which may be xe2x80x9csubstantially similarxe2x80x9d in nucleotide sequence, and which encode a molecule possessing similar activity to an hOAT polypeptide or fragment thereof. Permutations resulting from degeneracy of the genetic code are also considered functional derivatives.
A molecule is said to be xe2x80x9csubstantially similarxe2x80x9d to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
A xe2x80x9cchemical derivativexe2x80x9d of a polypeptide contains additional chemical moieties not normally a part of the polypeptide.
The term xe2x80x9cmammalianxe2x80x9d refers to such organisms as mice, rats, rabbits, goats, more preferably monkeys and apes, and most preferably humans.